Selection of suitable reference genes for normalization of RT-qPCR in three tissues of Northern bobwhite (Colinus virginianus) infected with eyeworm (Oxyspirura petrowi).

BACKGROUND: The Northern bobwhite (Colinus virginianus) is an economically important, and popular game bird in North America. Northern bobwhites have experiencing declines of > 3.5% annually in recent decades due to several factors. The eyeworm Oxyspirura petrowi is a nematode parasite frequently found in the eyes of bobwhites. Although reported frequently in wild bobwhites, there is no research to understand the host-parasite mechanism. Hence, it is important to investigate mechanisms of eyeworm invasion and immune modulation in bobwhite. Cytokine gene expression using RT-PCR is widely used to identify the innate immune response of a host to an infection. METHODOLOGY: In this study, we evaluated ten reference genes (HMBS, RPL19, RPL32, RPS7, RPS8, TATA, SDHA, YWHAZ, GAPDH, and ACTB) for their stability across three tissues (liver, spleen, and caecal tonsils) of control and O. petrowi infected Northern bobwhites. Primer efficiency and reference genes stability were assessed using GeNorm, NormFinder, and BestKeeper. RESULTS: Expression of these reference genes with respect to O. petrowi infection in bobwhites showed RPL32 and HMBS were the most stable genes in the liver, HMBS and SDHA were the most stable genes in the spleen, and HMBS and YWHAZ were equally stable reference genes in the caecal tonsils. CONCLUSION: Based on the geometric mean of all three analyses, our results indicate that the combination of RPL32 and HMBS for the liver, HMBS and SDHA for the spleen, and YWHAZ and HMBS for caecal tonsils might be used as reference genes for normalization in gene expression investigations on Northern bobwhites.

Emergence of Thelaziosis Caused by Thelazia callipaeda in Dogs and Cats, United States.

We report 2 autochthonous feline thelaziosis cases caused by the eyeworm Thelazia callipaeda and discuss the spread among dogs in the northeastern United States. Phylogenetic analysis suggests the parasite was introduced from Europe. Adopting a One Health approach is needed to limit further spread of T. callipaeda eyeworms in North America.

Efficacy of a combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) against Thelazia callipaeda in naturally infected cats.

This clinical study assessed the efficacy of a topical combination of esafoxolaner, eprinomectin and praziquantel (NexGard® Combo) in treating cats naturally infected with the eyeworm Thelazia callipaeda (Nematoda, Thelaziidae). On Study Day (SD) 0, sixteen client-owned cats with eyeworm infection were allocated to an untreated control group (G1, 8 cats) or to a NexGard® Combo treated group (G2, 8 cats) and subjected to ocular examination. Cats in G2 received the treatment as per label recommendations. On SD 7 and 14 (±1), cats were examined for the presence of eyeworms and clinical signs. On SD 14, eyeworms were collected and counted. On SD 7 and 14, all cats in G1 were still infected with eyeworms, while G2 cats were free from eyeworms on SD 7 and 14, demonstrating 100% efficacy (p < 0.0001). All collected eyeworms were morphologically and molecularly confirmed to be T. callipaeda. On SD 0, fifteen out of the sixteen cats (7 in G1 and 8 in G2) displayed inflammatory ocular signs. On SD 7, all eight untreated cats and seven treated cats displayed inflammatory ocular signs. On SD 14, five out of eight G2 treated cats had recovered, while the eight untreated cats still displayed inflammatory ocular signs. The treatment significantly reduced lacrimation and conjunctivitis (p = 0.0001). No adverse reactions occurred. This clinical study provides evidence that NexGard® Combo is highly safe and effective for the treatment of T. callipaeda infection in cats under field conditions.

Title: Efficacité d'une association d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) contre Thelazia callipaeda chez le chat naturellement infecté. Abstract: Cette étude clinique a évalué l'efficacité d'une association topique d'esafoxolaner, d'éprinomectine et de praziquantel (NexGard® Combo) dans le traitement des chats naturellement infectés par le ver oculaire Thelazia callipaeda (Nematoda, Thelaziidae). Au jour d'étude (JE) 0, seize chats appartenant à des clients et atteints d'une infection par le ver oculaire ont été attribués à un groupe témoin non traité (G1, 8 chats) ou à un groupe traité NexGard® Combo (G2, 8 chats) et soumis à un examen oculaire. Les chats du groupe G2 ont reçu le traitement conformément aux recommandations de l'étiquette. Aux JE 7 et 14 (±1), les chats ont été examinés pour détecter la présence de vers oculaires et de signes cliniques. Au JE 14, les vers oculaires ont été collectés et comptés. Aux JE 7 et 14, tous les chats du G1 étaient toujours infectés par des vers oculaires, tandis que les chats du G2 étaient exempts de vers oculaires aux JE 7 et 14, démontrant une efficacité de 100 % (p < 0,0001). Tous les vers oculaires collectés ont été confirmés morphologiquement et moléculairement comme étant T. callipaeda. Au JE 0, quinze chats sur seize (7 en G1 et 8 en G2) présentaient des signes oculaires inflammatoires. Au JE 7, les huit chats non traités et les sept chats traités présentaient des signes oculaires inflammatoires. Au JE 14, cinq des huit chats traités par G2 s'étaient rétablis tandis que les huit chats non traités présentaient toujours des signes oculaires inflammatoires. Le traitement a réduit de manière significative le larmoiement et la conjonctivite (p = 0,0001). Aucun effet indésirable n'est survenu. Cette étude clinique indique que NexGard® Combo est hautement sûr et efficace pour le traitement de l'infection à T. callipaeda chez les chats dans des conditions de terrain.

Molecular characterization of a novel Spiruromorpha species in wild Chinese pangolin by mitogenome sequence analysis.

Pangolins are susceptible to a variety of gastrointestinal nematodes due to their burrowing lifestyle and feeding habits, and few parasitic nematodes have been reported. Here, a Chinese pangolin with old wounds on its leg and tail was rescued from the Heyuan City, Guangdong Province. The cox1 and SSU rRNA of the worms from the intestine of the Chinese pangolin had the highest sequence identity of 89.58% and 97.95% to the species in the infraorder Spiruromorpha. The complete mitogenome of the worm was further assembled by next-generation sequencing, with a size of 13,708 bp and a GC content of 25.6%. The worm mitogenome had the highest sequence identity of 78.56% to that of Spirocerca lupi, sharing the same gene arrangement with S. lupi and some species in other families under Spiruromorpha. However, the mitogenome between the worm and S. lupi showed differences in codon usage of PCGs, sequences of NCR, and tRNA secondary structures. Phylogenetic analysis showed that the worm mitogenome was clustered with S. lupi in the family Thelaziidae to form a separate branch. However, it is still difficult to identify the worm in the family Thelaziidae because the species in the family Thelaziidae are confused, specifically S. lupi and Thelazia callipaeda in the family Thelaziidae were separated and grouped with species from other families. Thus, the parasitic nematode from the Chinese pangolin may be a novel species in Spiruromorpha and closely related to S. lupi. This study enriches the data on gastrointestinal nematodes in the Chinese pangolin.

IMPLICATIONS OF TREATING PARASITIC INFECTION IN NORTHERN BOBWHITE (COLINUS VIRGINIANUS) ON OVERALL HELMINTH LIFE CYCLE.

This study was conducted to determine how reducing the parasite burden in a definitive host may affect the prevalence in intermediate hosts. Here we used the eyeworm Oxyspirura petrowi and cecal worm Aulonocephalus pennula as model species. Northern bobwhite quail (Colinus virginianus) were provided an anthelmintic medicated feed in wild systems because of convincing evidence that these parasites were suppressing their populations. Eyeworm and cecal worm prevalence were measured in Orthopterans, which act as intermediate hosts, using polymerase chain reaction. Individuals were collected from a control site, a site treated for 2 yr, and a site treated for 5 yr. Orthopteran community composition was significantly different among the sites; however, an interaction between subfamily and site was not significant for the eyeworm. There was a significant reduction in eyeworm-infected Orthopterans on the 5-yr site compared with the other 2, suggesting that treatment of a definitive host may indeed affect the prevalence of eyeworms during other life-cycle stages. There was an interaction between the Orthopteran subfamily and the site for the cecal worm, so results were analyzed within each subfamily. A significant reduction in the prevalence of cecal worms was only found in the Cyrtacanthacridinae subfamily on the 5-yr site when compared with the other sites. However, the greatest prevalence in the Gomphocerinae and Oedipodinae subfamilies across all 3 sites was 4.1%. This indicates an unknown degree of cecal worm host specificity. Therefore, conclusions could not be made through the simple assessment of prevalence.

Social media as a tool for detecting underdiagnosed parasitic infections: the case of spirocercosis.

Spirocercosis is a parasitic disease caused by the nematode Spirocerca lupi that affects several canids around the world. In Costa Rica, few spirocercosis cases have been reported; however, it is suspected that the infection is currently underdiagnosed. As seen during the COVID-19 pandemic, social media has sped up the spread of scientific knowledge, raising practioner's awareness of the epidemiology and signs and manifestations of infections. The aim of this work was to use social media as a tool for increasing awareness of spirocercosis among veterinarians and investigate the frequency of cases occurring in Costa Rica. Infographic material about the infection was disseminated by Whatsapp, Facebook, and Instagram from January 2021 to July 2022 using a public account of the Laboratory of Helminthology of the University of Costa Rica. In addition, Google Forms questionnaires were completed by veterinarians to report the number of spirocercosis cases in their clinics, associated manifestations, diagnosis, treatment, and outcome of the disease. Remarkably, since 2020, a six-fold increase in the number of cases per year was noted. Clinical manifestations matched those reported elsewhere, and one of every three dogs infected with S. lupi died spontaneously or required euthanasia because of the infection. This work suggests that S. lupi infection in Costa Rica may have been underdiagnosed in the last decade, and the knowledge and awareness of clinical personnel play a major role in the detection of this and other infectious diseases.

[Epidemiological and clinical characteristics of human ocular helaziasis in China from 2011 to 2022 based on bibliometrics].

OBJECTIVE: To understand the clinical and epidemiological characteristics of human ocular thelaziasis patients in China. METHODS: Case reports regarding human ocular thelaziasis cases in China were retrieved in international and national electronic databases, including CNKI, VIP, CBM, Traditional Chinese Medical Literature Analysis and Retrieval System, Wanfang Database, PubMed and Web of Science from 2011 to 2022. Patients' gender, age, clinical symptoms, treatment, recurrence, site of infections, time of onset, affected eye, affected sites, number of infected Thelazia callipaeda, sex of T. callipaeda and source of infections were extracted for descriptive analyses. RESULTS: A total of 85 eligible publications were included, covering 101 cases of human ocular thelaziasis, including 57 males (56.44%) and 44 females (43.56%) and aged from 3 months to 85 years. The main clinical manifestations included foreign body sensation (56 case-times, 22.49%), eye itching (38 case-times, 15.26%), abnormal or increased secretions (36 case-times, 14.46%), tears (28 case-times, 11.24%) and eye redness (28 case-times, 11.24%), and conjunctival congestion (50 case-times, 41.67%) was the most common clinical sign. The most common main treatment (99/101, 98.02%) was removal of parasites from eyes using ophthalmic forceps, followed by administration with ofloxacin and pranoprofen. In publications presenting thelaziasis recurrence, there were 90 cases without recurrence (97.83%) and 2 cases with recurrence (2.17%). Of all cases, 51.96% were reported in four provinces of Hubei, Shandong, Sichuan, Hebei and Henan, and ocular thelaziasis predominantly occurred in summer (42.19%) and autumn (42.19%). In addition, 56.45% (35/62) had a contact with dogs. CONCLUSIONS: The human thelaziasis cases mainly occur in the continental monsoon and subtropical monsoon climate areas such as the Yellow River and the Yangtze River basin, and people of all ages and genders have the disease, with complex clinical symptoms and signs. Personal hygiene is required during the contact with dogs, cats and other animals, and individual protection is required during outdoor activities to prevent thelaziasis.

First report and molecular characterisation of Rhabdochona hospeti Thapar, 1950 (Thelazioidea: Rhabdochonidae) from tor barb, Tor tor (Hamilton) (Cyprinidae), in Assam, India.

Tor barbs, Tor tor (Hamilton), a cyprinid fish collected from Barak Valley, Assam, India were examined for helminth parasites. A nematode species of the genus Rhabdochona Railliet, 1916 (Rhabdochonidae) was found in the intestine of the fish specimens. The morphology of these nematodes was characterised by the presence of a funnel shaped prostom, head with four submedian sublabia, four cephalic papillae, long and slender posterior glandular oesophagus in both sexes, presence of 14 caudal papillae and two unequal spicules with pointed proximal tips in male specimens, and prominent post-equatorial vulval position, posteriorly directed vagina, uterus with fully scattered eggs in female specimens, and a tail pointed with a small mucron in both sexes. This nematode species was subsequently identified as Rhabdochona hospeti Thapar, 1950, reported herein for the first time in the northeastern India. In addition to its morphological description based on light microscopy, partial 18S and 28S rDNA sequences were generated to assess phylogenetic relationships of R. hospeti with other species of Rhabdochona.

Health impacts of gastrointestinal and ocular parasites in northern bobwhite (Colinus virginianus) in western Oklahoma, USA.

The northern bobwhite (Colinus virginianus) is a popular upland game bird that is suffering from severe and ongoing population decline. In this study, we investigated the potential health impacts of gastrointestinal and periorbital parasites in bobwhite in western Oklahoma, USA. A sample of 206 bobwhites from 2018 to 2020 indicated a low prevalence and diversity of parasites. However, at least one gastrointestinal or ocular parasite species was detected in 112 bobwhite (54.4%). A total of three gastrointestinal parasite species were detected, including Aulonocephalus pennula (54% prevalence, mean intensity 71.6 ± 99.8), Raillietina spp. (7%, 4.2 ± 1.9), and a single immature Mediorhynchus sp. acanthocephalan (0.5%). Burdens of A. pennula infections were negatively associated with fat stores in their bobwhite host. Low intensities (range 1-10, mean 3.9 ± 2.9) of eyeworms (Oxyspirura petrowi) were observed in 12.6% (26/206) of bobwhite sampled and were not associated with fat stores. No significant histologic lesions were associated with O. petrowi worms in ocular and surrounding tissues of 68 quail eyes examined, of which 26 (38%) were positive for eyeworms. Overall, the prevalence and intensity of parasites in bobwhite in Oklahoma were lower than in previous studies in Texas in similar physiographic regions. However, continued studies on the impacts of these parasites on quail health are needed as environmental and climate changes could alter the ecology and significance of these parasites.

Diversity of Thelazia spp. in domestic cattle from Romania: epidemiology and molecular diagnosis by a novel multiplex PCR.

BACKGROUND: Thelaziosis is a neglected vector-borne disease caused by parasitic nematode worms of the genus Thelazia which affects various hosts. Limited attention has been given to ungulate-associated Thelazia species. Current diagnosis of thelaziosis and the identification/differentiation of species heavily relies on morphological features. Therefore, we conducted an epidemiological study in Romanian cattle, with the aim to obtain morphological and molecular data that can be used for species identification. METHODS: The eyes of 705 slaughtered cattle were sampled and subjected to morphological identification, morphometric analysis, and molecular characterization. PCR amplification and sequence analysis were performed based on the cytochromec oxidase subunit 1 (COI) gene. Statistical tests assessed the correlations between infection parameters and ecological or biogeographical factors. A novel PCR method was developed based on the consensus sequence from each species. Specific forward primers were designed for each of the three species, and a reverse primer (COIintR) was used for all reactions. A consensus thermal profile was established by gradient PCR amplification of each species separately. RESULTS: Of the sampled cattle, 19.3% were infected with Thelazia spp. Prevalence varied significantly with ecogeographical factors. A total of 585 Thelazia nematodes were recovered, with T. rhodesi being the most abundant, followed by T. skrjabini and T. gulosa. Morphometric and molecular analyses supported the morphological identification, yielding unique sequences for each species. From the 59 T. rhodesi specimens sequenced, 29 unique sequences were obtained, with a 99.1-99.85% nucleotide identity to the only other COI sequence present in GenBank®. All nine T. gulosa isolates were unique (99.37-100% nucleotide identity to other sequences), while T. skrjabini specimens displayed 98.47-100% nucleotide identity to the sole available sequence. CONCLUSIONS: Bovine thelaziosis is prevalent in Romania, raising concerns for animal welfare and potential economic impacts. Infected cattle grazing alongside vulnerable wild ruminants, such as the European bison, may affect conservation efforts. Our newly developed multiplex PCR shows promise as a valuable surveillance tool, enabling the detection of occult infections in apparently healthy animals through lachrymal secretion testing.

EXPLORING A PLANT-DIVERSITY HYPOTHESIS TO EXPLAIN HELMINTH PREVALENCE IN NORTHERN BOBWHITE (COLINUS VIRGINIANUS) IN TEXAS, USA.

Helminths, in particular eyeworms (Oxyspirura petrowi) and cecal worms (Aulonocephalus pennula), may be a factor influencing northern bobwhite (Colinus virginianus) populations in Texas. Previous research has shown a discrepancy in helminth infections between the Rolling Plains and Rio Grande Plains of Texas, US, potentially caused by differences in intermediate host distribution and abundance. We explored an alternative hypothesis centered on plant diversity, given that many plants possess phytochemicals with anthelmintic properties. We predicted that plant diversity would be greater and bobwhite diet more diverse in the Rio Grande Plains than the Rolling Plains, which in turn would potentially expose bobwhites to more plants with anthelmintic properties and therefore result in lower parasite prevalence and intensity. We conducted a literature review of plant diversity, anthelmintic plants, and bobwhite diet in Texas to explore this hypothesis. We also quantified the relationship between helminth prevalence in bobwhites and latitude. We documented trends for higher plant species richness, greater number of anthelmintic plants, and more diverse bobwhite diet in the Rio Grande Plains compared to the Rolling Plains. In addition, we documented a trend for increasing helminth prevalence with latitude for eyeworms but not cecal worms. Our study provides circumstantial evidence supporting the plant-diversity hypothesis and warrants experimental testing.

Eight cases of canine thelaziosis found in two localities in Korea.

Thelazia callipaeda, a parasitic nematode that causes thelaziosis in various mammals, including humans, is known to be endemic in Korea. However, life cycle-related information on the parasite, primarily from human infection and a few dog cases, is limited. This study reports additional cases of T. callipaeda infections in dogs from both rural and urban areas in Korea, indicating the potential for transmission to humans and other animals. We collected 61 worms from 8 infected dogs from Paju and Cheongju Cities and observed their morphological characteristics under a light microscope. The findings indicate that T. callipaeda infections in animals in Korea may be underestimated and are distributed close to human environments. Our results contribute to the growing knowledge of the reservoir hosts of T. callipaeda in Korea and highlight the importance of continued surveillance and research to prevent and control this emerging zoonotic disease.

Occurrence of Thelazia callipaeda and its vector Phortica variegata in Austria and South Tyrol, Italy, and a global comparison by phylogenetic network analysis.

The zoonotic nematode Thelazia callipaeda infects the eyes of domestic and wild animals and uses canids as primary hosts. It was originally described in Asia, but in the last 20 years it has been reported in many European countries, where it is mainly transmitted by the drosophilid fruit fly Phortica variegata. We report the autochthonous occurrence of T. callipaeda and its vector P. variegata in Austria. Nematodes were collected from clinical cases and fruit flies were caught using traps, netting, and from the conjunctival sac of one dog. Fruit flies and nematodes were morphologically identified and a section of the mitochondrial cytochrome c oxidase subunit I gene (COI) was analysed. A DNA haplotype network was calculated to visualize the relation of the obtained COI sequences to published sequences. Additionally, Phortica spp. were screened for the presence of DNA of T. callipaeda by polymerase chain reaction. Thelazia callipaeda and P. variegata were identified in Burgenland, Lower Austria, and Styria. Thelazia callipaeda was also documented in Vienna and P. variegata in Upper Austria and South Tyrol, Italy. All T. callipaeda corresponded to haplotype 1. Twenty-two different haplotypes of P. variegata were identified in the fruit flies. One sequence was distinctly different from those of Phortica variegata and was more closely related to those of Phortica chi and Phortica okadai. Thelazia callipaeda could not be detected in any of the Phortica specimens.

MOLECULAR IDENTIFICATION OF INSECT INTERMEDIATE HOSTS WITH POTENTIAL PARASITE TRANSMISSION TO THE DEFINITIVE HOST NORTHERN BOBWHITE (COLINUS VIRGINIANUS).

Grassland birds have been declining substantially for the past several years. Habitat loss, degradation, and fragmentation as well as climate change are all thought to be the main drivers of the decline. However, as the declines continue to accelerate, it is becoming imperative to examine other factors that may contribute to population fluctuations. The nematodes Oxyspirura petrowi, Aulonocephalus pennula, and Physaloptera sp. are commonly found infecting northern bobwhite (Colinus virginianus), a game species of economic importance, and all 3 nematodes use insects as an intermediate host. Here we used polymerase chain reaction techniques to determine the occurrence of the 3 nematodes in 7 insect orders to uncover epidemiological patterns of the greatest potential for transmission to northern bobwhite. Insects were collected from March through September using sweep nets and pitfall traps. An R × C chi-squared test with Monte Carlo simulation was used to determine differences in the occurrence of the parasites across taxa and time. The results of the statistical analysis showed the nematodes are predominantly found in the order Orthoptera, and A. pennula and Physaloptera sp. showed epidemiological patterns in insects. However, no such pattern was observed with O. petrowi. An explanation for the lack of epidemiological pattern in O. petrowi is proposed and the diversity of known insect hosts of the 3 nematodes is increased.

Subconjunctival Thelazia in a Chinese Woman Aged 49 Years.

This case report describes a diagnosis of subconjunctival Thelazia callipaeda in a 49-year-old Chinese woman who presented with pruritis and a crawling sensation of the left eye for 1 week.

Ocular thelaziosis: an emerging human parasitosis in Europe.

Ocular thelaziosis is a parasitosis distributed mainly in East Asia, but increasingly described in Europe in different domestic and wild animals, including dogs, different wild canids and lagomorphs, and exceptionally in humans. In Spain, in some areas, a high infection prevalence rate has been described in domestic canids, which may lead to an increase in human cases. However, the description of human cases is still exceptional, which suggests that they are probably underdiagnosed. A case of ocular thelaziosis in a 2-year-old girl from an urban environment is described.

Spirocerca lupi in the stomach of two Andean foxes (Lycalopex culpaeus) from Chile.

The genus Spirocerca includes nematodes that parasitize the stomach and the oesophagus of carnivores, chiefly canids. Herein, we provide new data about the morphological, histopathological, and molecular characterization of Spirocerca sp. in Andean foxes (Lycalopex culpaeus) in Chile. Intact immature worms, identified as Spirocerca sp., were recovered in the lumen of the stomach from two foxes. Histologically, worms morphologically consistent with spirurid nematodes were present within the wall of the stomach and surrounded by nodular areas of inflammation with central necrotic debris. Molecular analysis of the cox1 gene yielded 19 sequences and 5 nucleotide sequence types with 99.95 to 99.98% similarity, being shared between both foxes. Nucleotide similarity ranged from 93.1 (with genotype 2 of S. lupi and S. vulpis) to 95.8% (with genotype 1 of S. lupi), a higher similarity than noted from sequences of S. lupi from an Andean fox from Peru (91.0 to 93.3%). However, the Poisson Tree Processes for species delineation did not support the existence of a new species Spirocerca. Phylogenetic and nucleotide analyses suggest that these specimens belong to a new variant or genotype of S. lupi or to a cryptic species. Whether the presence of the worms in the stomach has to do with genotypic differences in parasites or host or some combination is uncertain. Spirocerca lupi has never been found in Chilean dogs and must be investigated.

Validation of an LC-MS/MS method for assessment of fenbendazole sulfone drug residue in northern bobwhite liver.

The Northern Bobwhite (Colinus virginianus) is an economically important game bird within the Rolling Plains Ecoregion. Within this region, bobwhite is experiencing extreme cyclic population fluctuations which are resulting in a net decline in total population. It is suspected that within this region two helminth parasites, an eyeworm (Oxyspirura petrowi) and a cecal worm (Aulonocephalus pennula), are contributing to this phenomenon. However, this has been difficult to study as the primary mode of investigation would be the deployment of anthelmintic treatment. Unfortunately, no registered treatments for wild bobwhite currently exist. Thus, utilizing an anthelmintictreatment for wild bobwhite would require registration of that treatment with the U.S. Food and Drug Administration (FDA). As bobwhite are game birds that are hunted, they are considered food-producing animals to the FDA, and as such require the assessment for the withdrawal of the drug residues to be assessed for human food safety. In this study, we optimized and validated a bioanalytical method for the quantification of fenbendazole sulfone in bobwhite following the U.S. FDA Center for Veterinary Medicine Guidance for Industry #208 [VICH GL 49 (R)] for assessment of fenbendazole sulfone drug residue in Northern bobwhite liver. The official method for quantifying fenbendazole sulfone in domestic chicken (Gallus gallus) was adapted for use in bobwhite. The validated method quantitation range is 2.5-30 ng/mL for fenbendazole with an average recovery of 89.9% in bobwhite liver.

First report of Thelazia callipaeda Railliet & Henry, 1910 in a zoo lion (Panthera leo) (Linnaeus, 1758) from Romania.

Thelazia callipaeda, the zoonotic "oriental eye worm", is an emerging nematode known to infect a wide range of hosts, particularly carnivores (wild and domestic canids and felids, mustelids, and ursids), but also other mammal groups (suids, lagomorphs, monkeys, and humans), across a wide geographical area. New host-parasite associations and human cases have mostly been reported in endemic areas. A less studied group of hosts is represented by zoo animals that may harbor T. callipaeda.A 23-year old male lion (Panthera leo) that was born in a public zoo in Reșița (Caraș-Severin County, Romania) was submitted to the University of Agricultural Sciences and Veterinary Medicine of Cluj-Napoca for investigations after death from unknown reasons in November 2022. Four nematodes were collected from the right eye during the necropsy and submitted to morphological and molecular characterization.Collected nematodes were morphologically identified as three females and one male T. callipaeda. The BLAST analysis revealed 100% nucleotide identity to numerous isolates of T. callipaeda haplotype 1.The present paper presents a new host-parasite association between T. callipaeda and P. leo from a zoo in Romania.